线粒体抗病毒信号蛋白真核表达载体的构建及鉴定  

Construction and identification of mitochondrial antiviral signaling protein in eukaryotic expression vector

作　　者：刘翠玉[1] 李玉军[1] 孙岚[1] 纪羽婷[1] 张庆猛[1] 魏兰兰[1] 张凤民[1] 

机构地区：[1]哈尔滨医科大学医学微生物学教研室,黑龙江省感染与免疫重点实验室,黑龙江省感染与免疫创新团队,黑龙江省普通高校病原学重点实验室,150081

出　　处：《国际免疫学杂志》2015年第2期120-123,共4页International Journal of Immunology

基　　金：基金项目：黑龙江省自然科学基金（QC2014C088）;黑龙江省卫生和计划生育委员会科研课题（2012-759）

摘　　要：目的构建线粒体抗病毒信号蛋白真核表达载体。方法通过聚合酶链反应（PCR）的方法扩增获得线粒体抗病毒信号蛋白基因的完整序列，进而纯化回收后连接到pMD^TM18-T载体上，将pcDNA6／myc．HisA载体和带有目的片段的pMD^TM18-T载体同时进行双酶切，酶切产物过夜连接转化至大肠杆菌JMl09，挑取阳性克隆pcDNA6／myc—HisA—MAVS扩增后，提取重组质粒；利用PCR和核酸测序验证线粒体抗病毒信号蛋白真核表达载体构建的正确性；应用Western blotting的方法检测融合蛋白的表达。结果线粒体抗病毒信号蛋白真核表达载体构建成功，并且该载体能够在OL细胞中表达融合蛋白。结论成功构建线粒体抗病毒信号蛋白真核表达载体，为进一步研究该蛋白的功能和作用机制奠定基础。Objective To construct the eukaryotic expression vector of mitochondrial antiviral signaling protein. Methods The target DNA fragments of mitochondrial antiviral signaling protein gene was obtained from PCR amplification, then the cDNA fragment was ligated into pMDTM 18-T Vector. After double-enzyme digestion, the MAVS gene was transferred into the eukaryotic expression vector pcDNA6/myc-HisA. The constructs transformed into E. coil JM109 and positive clones were picked by peDNA6/myc-HisA-MAVS PCR am- plification. This construct was confirmed by PCR and DNA sequencing. Results The eukaryotic expression vector of mitochondrial antiviral signaling protein was constructed correctly. The recombinant vector was transfected into OL cells, and the expression of the recombinant protein was detected by western blotting. Conclusion The successfully constructed pcDNA6/myc-HisA MAVS plasmid is useful to study the gene＇ s function and effects in cells. This study is for further study of the mechanism of gene function.

关 键 词：线粒体抗病毒信号蛋白 真核表达载体 序列分析 

分 类 号：R440[医药卫生—诊断学]